Carbohydrates of influenza virus hemagglutinin - American Chemical

Immunol. 17, 1443-1448. Carbohydrates of Influenza Virus Hemagglutinin: Structures of the Whole. Neutral Sugar Chains1-. Akira Matsumoto, Hideo Yoshim...
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Biochemistry 1983, 22, 188-196

tyrosine at residue 16. Consequently, the most likely explanation for this observation is the coisolation of the /3 chains of HLA-DR antigens with that of a structurally very similar human Ia antigen that is phenotypically distinct from HLADR. The two regions of high polymorphism in the /3 chains, residues 7-13 and 26-31, are of adequate length to represent distinct antigenic sites since it has been reported that a hexapeptide is approximately the size of an antigenic determinant (Atassi, 1975). We are currently synthesizing these two regions of the molecule and are attempting to produce both polyclonal and monoclonal antibodies reactive with them to determine whether these two regions of amino acid sequence variability are important for the recognition of allospecific sites. The seemingly high degree of structural polymorphism that exists among the @ chains of HLA-DR1 and HLA-DR2 antigens is somewhat surprising in light of a report by Kratzin et al. (1981), who showed that an antigen preparation isolated from a homozygous B lymphoid cell line (HLA-DR2,2) is actually comprised of a pool of seven /3 chains which are highly variable in only one five amino acid region (residues 65-69) of each molecule. If, as suggested by Kratzin et al. (1981), these @ chains are indeed the product of a cluster of different genes, then the structural difference between two HLA-DR allospecificities (HLA-DR1 and HLA-DR2) are more numerous than those detected between HLA-DR and other human Ia gene products. Acknowledgments The excellent technical assistance of Gail Kelly and Karen Truesdell is gratefully acknowledged as is the skilled secretarial assistance of Bonnie Filiault.

References Allison, J. P., Walker, L. E., Russell, W. A., Pellegrino, M. A., Ferrone, S.,Reisfeld, R. A., Frelinger, J. A., & Silver, J. (1978) Proc. Natl. Acad. Sci. U.S.A. 7 5 , 3953-3956. Amos, D. B., & Kostyu, D. D. (1980) Advances in Human Genetics (Harris & Hischorn, Eds.) p 137, Plenum Press, New York. Atassi, M. Z. (1975) Immunochemistry 12, 741-744. Freed, J. H. (1980) Mol. Zmmunol. 17, 453-462. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., & Dreyer, W. J. (1981) J . Biol. Chem. 256, 7990-7997. Hunkapiller, M. W., & Hood, L. E. (1980) Science (Washington, D.C.) 207, 523-525. Kaufman, J. F., Andersen, R. L., & Strominger, J. L. (1981) J . Exp. Med. 152, 37s-53s. Kratzin, H., Yang, C., Gotz, H., Pauly, E., Kolbel, S.,Egert, G., Thinnes, F. P., Wernet, P., Altevogt, P., & Hilschmann, N. (1981) Hoppe-Seyler's 2. Physiol. Chem. 362, 1665-1 669. Mahoney, W. C., & Hermodson, M. A. (1979) Biochemistry 18, 3810-3814. Silver, J., & Ferrone, S. (1979) Nature (London) 279, 436-438. Springer, T., Kaufman, J., Terhorst, C., & Strominger, J. (1978) Zr Genes and Za Antigens (McDevitt, H. O . , Ed.) pp 229-234, Academic Press, New York. Tosi, R., Tanayaki, R., Centes, D., Ferrara, G. B., & Pressman, D. (1978) J . Exp. Med., 148, 1592-1602. Walker, L. E., & Reisfeld, R. A. (1982) J . Biol. Chem. 257, 7940-7943. Walker, L. E., Ferrone, S., Pellegrino, M, A,, & Reisfeld, R. A. (1980) Mol. Immunol. 17, 1443-1448.

Carbohydrates of Influenza Virus Hemagglutinin: Structures of the Whole Neutral Sugar Chains? Akira Matsumoto, Hideo Yoshima, and Akira Kobata*

ABSTRACT:

The carbohydrates of BHA, a solubilized hemagglutinin of influenza virus by bromelain digestion, were quantitatively released as oligosaccharides by hydrazinolysis. The oligosaccharide mixture was separated into a neutral and two acidic fractions by paper electrophoresis. Both acidic fractions were resistant to sialidase digestion but were slowly converted to the neutral fraction by incubation with sulfatases. The neutral fraction which comprised about 80% in molar ratio of total oligosaccharides was separated into 13 oligosaccharides

by paper chromatography and by Con A-Sepharose column chromatography. Structural studies of these oligosaccharides by sequential exoglycosidase digestion and by methylation analysis revealed that BHA contains a series of high mannose type and bi-, tri-, and tetraantennary complex type sugar chains. Occurrence of Gal@l-+3GlcNAc outer chain in two and bisectional N-acetylglucosamine in one of the biantennary sugar chains is an interesting characteristic of the sugar chains of BHA.

A m o n g the proteins coded by influenza virus, the hemagglutinin and the neuraminidase are integral membrane

glycoproteins which construct spikes on the viral envelope. When hemagglutinin (HA) binds specifically to the sialated components of host cell plasma membranes, HA adsorbs virus particles to the cell surface and induces penetration of viral RNA into the cell by fusing the viral envelope with the cellular membrane. The hemagglutinin is also the major surface antigen of the virus, and variations in its antigenic structure accompany the recurrences of influenza epidemics in man. The HA of A2/Hong Kong/1968 virus is a trimer of molecular weight of about 220000 (Wiley & Skehel, 1977), and each subunit of the glycoprotein is composed of two di-

From the Department of Biochemistry, Kobe University School of Medicine, Chuo-ku, Kobe, Japan. Receiued August 3, 1982. This work has been supported in part by Grant-in-Aids for Scientific Research, the Ministry of Education, Science and Culture of Japan, and by a research grant from Yamanouchi Foundation for Research on Metabolic Disorders. *Address correspondence to this author at the Department of Biochemistry, Institute of Medical Science, Tokyo University, 4-6- 1 Shirokanedai, Minato-ku, Tokyo, Japan.

0006-2960/83/0422-0188$01.50/0

0 1983 American Chemical Society

VOL. 22, NO. 1 , 1983

CARBOHYDRATES OF INFLUENZA VIRUS

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GlcNAc@1-4(GlcNAc@1-2)Mana 1-6(GlcNAc@ 1+2Mana l-3)ManO 1-4GlcNAcP 1-4( Fuca 1-6)GlcNAcoT (GlcNAc3-Man3~GlcNAc.Fuc~GlcNAcoT) and GlcNAc@l-4(GlcNAc@1-2)Mana 1-6 [GlcNAc/3l-6([email protected])Mana 1-31 Man@1-4GlcNAca 1+4( Fuca 1-6)GlcNAcoT (GlcNAc4.Man3.GlcNAcoT) were obtained by jack bean 0galactosidase digestion of Ga13~GlcNAc3.Man3.G1cNAc. F u c * G ~ c N A c ~and T Gal,~GlcNAc4~Man3~G1cNAc~Fuc~ GlcNAcoT, respectively. Gal@l-+4GlcNAc@l-2Manal-6(Gal@l-4GlcNAc@ 1-2Mana 1-+3) (GlcNAc@l-+4)Man@l-+4GlcNAc@l+4(Fucal+6)GlcNAcoT (Gal,. Experimental Procedures GlcNAc2~GlcNAc-Man3~GlcNAc~Fuc~GlcNAcoT) and Preparation of Bromelain-Released Hemagglutinin (BHA). G l c N A c @ l + 2 M a n a 1+6(GlcNAc@ 1+ 2 M a n a 1-3)BHA was obtained by bromelain digestion of X-3 1 strain of (GlcNAc@1-4)Manp 1-4GlcNAcP 1+4( Fuca 1-6)A2/Hong Kong/ 1968 virus and purified according to the GlcNAcm (GlcNAc2~GlcNAc.Man3~GlcNAc.Fuc.GlcNA~T) method of Brand & Skehel (1972). As reported previously were obtained from glycophorin A (Yoshima et al., 1980a). (Skehel & Waterfield, 1975), the BHA contains all carbohGal@1-4( Fuca 1-3)GlcNAc@ 1-2Mana 1-6 [Gal@1-+4ydrate moiety of intact HA and consists of two types of gly(Fuca 1-3)GlcNAcP 1-2Mana 1-31 Man@1-4GlcNAccopeptide of apparent molecular weights 58 000, HA1, and (Gal,.Fuc2.GlcNAc2-Man3@l-4(Fucal-6)GlcNAcoT 21 000, HA2, which can be separated by sodium dodecyl G~cNA~.Fuc.GIcNAc~T) and a mixture of Gal@l-4sulfate (NaDodS04)-polyacrylamidegel electrophoresis in the (Fucal-+3)GlcNAc@l-+2Mana1-(6- or -3)[Gal@l-presence of 0-mercaptoethanol. 4GlcNAcP 1-+2Mana1-(3- or -6)]Man@l-+4GlcNAc@l+Oligosaccharides. Mana 1-6( Mana 1-3) Mana 1-64(Fucal-.6)GlcNAcoT (Ga12.Fuc.GlcNAc2.Man3(Mana 1-3) Man@l-4GlcNAc@l-4GlcNAcoT (Man5. G ~ c N A ~ . F u ~ . G ~ c N Awere c ~ Tobtained ) from human parotid GlcNAc.GlcNAcoT),' (Mana 1~2)~Man5~GlcNAc~GlcNAcma-amylase (Yamashita et al., 1980b). (Man,.GlcNAc.GlcNAcoT), and (Manal-2),.Man5. Chemicals and Enzymes. NaB3H4 (246 mCi/mmol) was GlcNAc.GlcNAcm (Man,.GlcNAc.GlcNA*) were obtained purchased from New England Nuclear, Boston, MA. NaB2H4 by hydrazinolysis of bovine pancreatic ribonuclease B (Liang (98%) was purchased from Merck Co., Inc., Darmstadt, F. et al., 1980). Man@l-4GlcNAc@1-4GlcNAcoT (Man. R.G. @-Galactosidase, @-N-acetylhexosaminidase, and aGlcNAc.GlcNAcoT) and GlcNAc@l-4GlcNAcoT were obmannosidase were purified from jack bean meal by the method tained by sequential digestion of Man5~GlcNAc~GlcNAcoT of Li & Li (1972). @-Galactosidase and 0-N-acetylhexoswith jack bean a-mannosidase and 0-mannosidase, respectively. aminidase were also purified from the culture medium of N-Acetyl[3H]gluc~~aminitol was obtained by NaB3H4 reDiplococcus pneumoniae by the method of Glasgow et al. duction of N-acetylglucosamine (Takasaki & Kobata, 1978). (1977). Galj31-.4GlcNAc@1+2Mana 1-6(Gal@1-+4GlcNAc@l-Diplococcal @-galactosidasehydrolyzes Gal@l-+4GlcNAc 2Mana1-3)Man@l-4GlcNAc@l-4(Fuca1-6)GlcNAcm linkage but not Galpl-3GlcNAc and Ga1/31-+6GlcNAc (Gal,~GlcNAc,~Man3~GlcNAc.Fuc.GlcNAcoT) was obtained linkages (Paulson et al., 1978). Diplococcal @-N-acetylfrom subcomponent C l q of the first component of human hexosaminidase also has a useful substrate specificity for the complement (Mizuochi et al., 1978). GlcNAc@l+structural study of asparagine-linked sugar chains (Yamashita 2 M a n a 1 -+6( G l c N A c @ l - + 2 M a n a l - 3 ) M a n @ l - et al., 1981): The enzyme hydrolyzes the GlcNAc@l-+2Man 4GlcNAcP 1-4( Fuca 1-6)GlcNAcoT (GlcNAc2.Man3. linkage but not GlcNAc@l-+4Man and GlcNAc@l-+6Man G~CNA~.FUOG~CNAC~T), Mana 1+6( Mana 1-+3)Man@l-linkages at low substrate concentration. The GlcNAc@l-4GlcNAc@l-4(Fuca 1-6)GlcNAcoT (Man,.GlcNAc.Fuc. 2Man linkage in the GlcNAc@l-4(GlcNAc@l-2)Man GlcNAcoT), Man@l-4GlcNAc@l-+4(Fuca 1-+6)GlcNAcoT group is cleaved by the enzyme, but the linkage in (Man.GlcNAc.Fuc.GlcNAcoT),and GlcNAc@1-+4(Fuca 1-GlcNAcP 1-6( GlcNAcP 1-2) Man group is not cleaved. 6)GlcNAcoT (G~CNAC.FU~.GICNAC~T) were obtained by sea-Mannosidase of Aspergillus saitoi was purified by the quential digestion of Ga12~GlcNAc2~Man3~G1cNAc-Fuc~ method of Ichishima et al. (1981). This enzyme hydrolyzes GlcNAcoT with jack bean @-galactosidase,jack bean @-Nthe Manal-2Man linkage but not Manal+3Man and acetylhexosaminidase,jack bean a-mannosidase, and @-manMana1-6Man linkages (Yamashita et al., 1980a) and is a nosidase, respectively. Fuca 1-6GlcNAcoT was isolated from useful reagent for identifying the high mannose type oligothe urine of fucosidosis patients (Nishigaki et al., 1978). saccharides (Mizuochi et al., 198 1). Snail @-mannosidase Mana 1-6(Gal@ 1-4GlcNAc@1-2Manal-3)Man@l-(Sugawara et al., 1972) and Charonia lampas a-fucosidase 4GlcNAcpl-4(Fucal-6)GlcNAcoT (Gal.GlcNAc.Man3. (Nishigaki et al., 1974) were kindly supplied by Seikagaku GIcNAc*Fuc.G~cNAc~T), Gal@1-4GlcNAc@l-4(Gal@l-Kogyo Co., Tokyo. a-Fucosidase I was purified from almond 4GlcNAcP 1+ 2 ) M a n a 1-6( Gal@1-4GlcNAcp 1 emulsin as reported previously (Yoshima et al., 1979). An2Mana1-3)Manp 1-+4GlcNAc@l+4(Fuca 1-+6)GlcNAcoT other a-fucosidase was purified from Bacillus fulminans ac(Ga13~GlcNAc3~Man3~GlcNAc.Fuc.GlcNAcoT), and Gal@l-cording to the method of Kochibe (1973). These fucosidases 4GlcNAcP 1-4 (Gal@1+4GlcNAc@ 1-2) M a n a 1-6have different substrate specificities: the Charonia lampas [Gal@1-4GlcNAc@l-6(Gal@l-4GlcNAc@l-2)Mana 1-enzyme hydrolyzes all a-fucosyl linkages so far reported, while 31 Man@l-4GlcNAc@l-4(Fucal-6)GlcNAcoT (Gal,. the Bacillus enzyme cleaves the Fuca 1-2Gal linkage only GlcNAc4-Man3.GlcNAc.Fuc.GlcNAcoT) were obtained from and the almond enzyme cleaves specifically the a-fucosyl calf thymocyte plasma membrane (Yoshima et al., 1980b). linkages in Gal@1-4( Fucal+3)GlcNAc-and Gal@1+3(Fucal-4)GlcNAc-groups. Sulfatases from limpets (type Subscript OT is used in this paper to indicate NaB3H,-reduced V) and abalone (type VII) were purchased from Sigma sugars. All sugars mentioned in this paper were of D configuration, Chemical Co., St. Louis, MO. except for fucose which had an L configuration.

sulfide-linked polypeptide chains called HA1 and HA2. The HA can be removed from virus particles by bromelain digestion, and the soluble glycoprotein (BHA) thus obtained lacks the membrane anchoring C-terminal peptide of the subunits (Skehel & Waterfield, 1975) and has been crystallized. The three-dimensional structure of BHA isolated from X-31 (H3N2) virus has recently been elucidated by X-ray crystallography (Wilson et al., 1981), and this paper will describe structural studies of the whole carbohydrate moieties of BHA obtained from the X-31 virus.

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MATSUMOTO, YOSHIMA, A N D KOBATA

BIOCH E M I STR Y

Analytical Methods. High-voltage paper electrophoresis was performed with either pyridine/acetate buffer, pH 5.4 (pyridine/acetic acid/water, 3:1:387), at a potential of 89 V/cm for 1.5 h, or 0.06 M borate buffer, pH 9.5, at 40 V/cm for 5.5 h. Descending paper chromatography was performed with ethyl acetate/pyridine/acetic acid/water (5:5: 1:3). Radioactivity was determined on an Aloka liquid scintillation spectrometer, Model LSC-700. Radiochromatoscanning was performed with a Packard radiochromatogram scanner, Model 7201. Bio-Gel P-4 (smaller than 400 mesh) column chromatography was performed as reported in the previous paper (Yamashita et al., 1982) by using a 2-m column. Methylation analysis of oligosaccharides was performed as described in the previous paper (Endo et al., 1979). Concanavalin A (Con A)-Sepharose column chromatography was performed as follows. A column (0.5 X 5 cm) of Con A-Sepharose was equilibrated with 0.01 M tris(hydroxymethy1)aminomethane hydrochloride (Tris-HC1) buffer, pH 7.5, containing 0.1 M NaCI. Radioactive oligosaccharides in less than 0.1 mL of the buffer were applied to the column, and the column was washed with 10 mL of the buffer. The column was then washed with 10 mL of the buffer containing 0.1 M methyl a-mannopyranoside. Fractions of 1 mL were collected, and the radioactivity in each tube was determined on an aliquot of sample by the liquid scintillation method. As reported by Ogata et al. (1975), complex-type sugar chains in which the two a-mannosyl residues of the trimannosyl core are either free or substituted only at the C-2 position by outer chains bind to the column and are eluted by 0.1 M methyl a-mannopyranoside solution. Periodate oxidation of radioactive oligosaccharides was performed as reported previously (Yoshima et al., 1981). Enzymatic Digestion of Oligosaccharides. Radioactive oligosaccharides [(l-2) X lo4 cpm] were incubated with one of the following mixtures at 37 OC for 18 h: (1) jack bean 0-galactosidase digestion, enzyme (0.7 unit) in 0.1 M citrate/phosphate buffer, pH 3.5 (50 pL); (2) jack bean P-Nacetylhexosaminidase digestion, enzyme (0.7 unit) in 0.1 M citrate/phosphate buffer, pH 5.0 (50 pL); (3) diplococcal P-galactosidase digestion, enzyme (20 milliunits) in 0.1 M citrate/phosphate buffer, pH 6.0 (50 pL); (4) diplococcal 0-N-acetylhexosaminidase digestion, enzyme (4 milliunits) in 0.1 M citrate/phosphate buffer, pH 6.0 (50 pL); (5) jack bean a-mannosidase digestion, enzyme (0.5 unit) in 0.1 M acetate buffer, pH 4.5 (50 pL); (6) Aspergillus a-mannosidase digestion, enzyme (0.15 pg) in 0.1 M acetate buffer, pH 5.0 (30 pL); (7) P-mannosidase digestion, enzyme (20 milliunits) in 0.5 M sodium citrate buffer, pH 4.5 (30 pL); (8) Charonia lampas a-fucosidase digestion, enzyme (0.2 unit) in 0.1 M acetate buffer, pH 4.0, containing 0.1 M NaCl (60 pL); (9) almond a-fucosidase I digestion, enzyme (40 microunits) in 0.1 M citrate/phosphate buffer, pH 5.0 (50 pL); (10) Bacillus a-fucosidase digestion, enzyme (17.5 pg) in 0.05 M sodium phosphate buffer, pH 6.6 (30 pL);(1 1 ) diplococcal P-galactosidase and diplococcal P-N-acetylhexosaminidasedigestion, P-galactosidase (20 milliunits) and P-N-acetylhexosaminidase (4 milliunits) in 0.1 M citrate/phosphate buffer, pH 6.0 (50 pL). Fifty microliters of toluene was added to all reaction mixtures to inhibit bacterial growth. Reactions were terminated by heating the reaction mixture in a boiling water bath for 2 min, and the products were analyzed by Bio-Gel P-4 column chromatography. Release of the Carbohydrate Moieties of BHA. BHA (37 mg) was suspended in 0.5 mL of anhydrous hydrazine and subjected to 10-h hydrazinolysis as reported in the previous

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DISTANCE FROM ORIGIN (cm) FIGURE 1: Paper electrophoretogram of oligosaccharides liberated from X-3 1 BHA glycoprotein by hydrazinolysis. After reduction with NaB3H,, the oligosaccharide mixture was subjected to paper electrophoresis at pH 5.4. Arrow indicates the position to which bromophenol blue migrated.

paper (Takasaki et al., 1982). The oligosaccharide fraction thus obtained was dissolved in 0.5 mL of 0.05 N NaOH. To one-fifth of the solution was added 10 nmol of lactose as an internal standard, and the sugars in the solution were reduced with NaB3H4(1.5 mCi) as reported previously (Takasaki & Kobata, 1974). [3H]Lactitol and tritium-labeled oligosaccharide fractions were separated by paper chromatography by using solvent I. The yield of total radioactive oligosaccharides was 2.2 X lo7 cpm. On the basis of the specific activity of the NaB3H4calculated from the radioactivity incorporated into lactitol and the molecular weight (79 000) of a BHA subunit, the sugar chains released from 1 mol of BHA was calculated as 6.5 mol. The remaining sample was reduced with 5 mg of NaB2H, to obtain deuterium-labeled oligosaccharide mixture. For facilitation of detection of oligosaccharides in further purification procedures, half of the tritium-labeled oligosaccharide mixture was added to the deuterium-labeled sample. Results Fractionation of Oligosaccharides by Paper Electrophoresis. The radioactive oligosaccharide fraction obtained from X-3 1 BHA glycoprotein by hydrazinolysis was spotted on Whatman 3MM paper and subjected to paper electrophoresis at pH 5.4. As shown in Figure 1, it was separated into a neutral (N) and two acidic (A1 and A2) fractions. These three fractions were recovered from paper by elution with water. The percent molar ratio of N, A l , and A2 calculated on the basis of their radioactivities was 79:16:5. Aliquots (1.5 X lo4 cpm) of N, Al, and A2 were hydrolyzed in 0.4 mL of 4 N HC1 at 100 OC for 2 h, and the reaction mixture was freed from HC1 by repeated evaporation with HzO. The hydrolysates were N-acetylated and analyzed by paper electrophoresis using borate buffer as reported previously (Takasaki & Kobata, 1978). N-Acetylglucosaminitol was the only radioactive component detected from the three fractions. Therefore, N-acetylglucosamine is located at the reducing termini of all oligosaccharides in the three fractions as expected from the hydrazinolysis reaction of asparagine-linked sugar chains (Takasaki et al., 1982). Both A1 and A2 were completely resistant to sialidase digestion. When AI and A2 were incubated with sulfatases, they were slowly converted to neutral components. These results indicated that the acidic nature of A1 and A2 cannot be ascribed to sialic acid like most other asparagine-linked sugar chains but to sulfate residues as suggested by Printer & Compans (1 975). Fractionation of Neutral Oligosaccharide by Paper Chromatography. When the N fraction was subjected to paper chromatography for 7 days, a chromatogram shown in Figure 2A was obtained. Fractions N3 and N4 as indicated by white bars in Figure 2A were recovered from paper by elution with H 2 0 and subjected to the same paper chromatography for 28

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C A R B O H Y D R A T E S OF I N F L U E N Z A V I R U S

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DISTANCE FROM ORIGIN (cm) Fractionation of neutral oligosaccharides by paper chromatography. (A) N fraction obtained by paper electrophoresis was subjected to descending paper chromatography by using solvent I for 7 days; (B) fraction N3 in (A) was subjected to rechromatography with the same solvent for 28 days; (C) fraction N4 in (A) was subjected to rechromatography with the same solvent for 28 days.

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FIGURE 2:

days. As shown in Figure 2B,C, N3 was separated into two (N3-1 and N3-2) and N4 into at least seven (N4-1-N4-7) fractions. The 11 fractions in total as indicated by black bars in Figure 2A-C were recovered from papers. The percent molar ratio of N1, N2, N3-1, N3-2, N4-1, N4-2, N4-3, N4-4, N4-5, N4-6, and N4-7 calculated on the basis of their radioactivities was 9.8:13.9:34.5:15.2:13.0:2.2:4.2:1.5:2.1:2.3:1.7. Structures of N1, N2, N3-I, and N3-2 Fractions. When N1, N2, N3-1, and N3-2 were analyzed by Bio-Gel P-4 column, they were eluted as single components. The mobilities of N1, N2, and N3-2 were the same as authentic Man5. GlcNAc.GlcNAcoT, Man,.GlcNAc-GlcNAcoT, and Man,. GlcNAoGlcNAcoT, respectively (Figure 3A). When N2 and N3-2 were incubated with Aspergillus a-mannosidase, they were converted to a radioactive component with the same mobility as authentic Man5-GlcNAc.GlcNAcoTreleasing one and two mannose residues, respectively (Figure 3C). The mobility of N3-1 in the Bio-Gel P-4 column was the same as that of authentic Man,-GlcNAc-GlcNAco,. However, this oligosaccharide was totally resistant to Aspergillus a-mannosidase digestion (data not shown). When incubated with jack bean @-N-acetylhexosaminidase, it was converted to a radioactive component with the same mobility as authentic Man5.GlcNAc.GlcNAcoT releasing an N-acetylglucosamine residue (Figure 3B). That the structures of the radioactive components in Figure 3C are Man5.GlcNAc.GlcNAcoT was confirmed by sequential exoglycosidase digestion: they were converted to Man-GlcNAc.GlcNAcoT and GlcNAc@l-+4GlcNAcoT by sequential digestion with jack bean a-mannosidase (Figure 3D) and @-mannosidase (Figure 3E), respectively, and finally converted to N-acetyl [3H]glucosaminitol by jack bean @-N-acetylhexosaminidasedigestion (Figure 3F). These results indicated that N1, N2, and N3-2 are a series of high mannose type sugar chains, and N3-1 is one of the processing intermediates as shown in Figure 8. Because of the limited amount of sample available, methylation analysis of these oligosaccharides was not performed. Structures of N4-I, N4-2, and N4-3 Fractions. When subjected to Con A-Sepharose column chromatography, 68% of N4-1 fraction bound to the column and eluted with methyl a-mannopyranoside solution, while N4-2 and N4-3 fractions did not bind to the column. The fraction of N4-1 bound to

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Sequential exoglycosidase digestion of oligosaccharide N1, N2, N3-1, and N3-2. Black arrows indicate the eluting positions of FIGURE 3:

glucose oligomers (numbers indicate the glucose units) and void volume (Vo). White arrows indicate the positions where authentic oligosaccharides eluted: I, Man,-GlcNAc.GlcNAcoT; 11, Man6. G~cNAoG~cNAcoT;111, Man,.GlcNAoGlcNAcoT; IV, Man.

GIcNAoGIcNAcoT; V, GlcNAc~l+4GlcNAcoT; VI, N-acetyl[3H]glucosaminitol. (A) Oligosaccharides N1 (-), N2 (---), and N3-1 or N3-2 (- --); (B) oligosaccharide N3-1 incubated with jack bean P-N-acetylhexosaminidase;( C ) oligosaccharides N2 and N3-2 incubated with Aspergillus a-mannosidase; (D) the radioactive peak in (B) or ( C ) incubated with jack bean a-mannosidase; (E) the

radioactive peak in (D) incubated with P-mannosidase; (F) the radioactive peak in (E) incubated with jack bean 0-N-acetylhexosaminidase.

the column will be called in this paper as N4-lb and that unbound as N4-lu. N4- 1b, N4-2, and N4-3 gave single peaks upon Bio-Gel P-4 column chromatography (Figure 4A). When incubated with jack bean @-galactosidase, 1 and 2 mol of galactose were released from N4-2 and N4-1 b, respectively, while N4-3 remained unchanged (Figure 4B). The radioactive products in Figure 4B from N4-2 and N4-lb released 1 and 2 mol of N-acetylglucosamine residue by jack bean @-N-acetylhexosaminidase digestion (Figure 4C), while N4-3 was still resistant to the enzyme digestion (data not shown). The mobility of the radioactive product from N4-lb at this stage was the same as that of authentic Man,~GlcNAc.Fuc~GlcNA~~~. That the component actually has the structure Mana1-+6(Manal--3)Man@l-+4GlcNAc@l-+4(Fucal--+6)GlcNAcoTwas confirmed by sequential exoglycosidase digestion documented in Figure 4D-G and methylation analysis in Table I. Therefore, the structure of N4-lb should be Gal@l-+GlcNAc@l-+M a n a 1-+6(Gal@1-+GlcNAc@l-+Mana 1-+3)Man@l-+4GlcNAc@l44(Fuca 1+6)GlcNAcoT. That the mobility of N4-lb in Bio-Gel P-4 column chromatography was the same as that of authentic Ga12~GlcNAc2~Man3~G1cNAc~Fuc~ GlcNAcoT (Figure 4A) also supports this assumption. The mobilities of N4-2 and N4-3 in Bio-Gel P-4 column were the same as those of authentic Ga12.Fuc.GlcNAc2. Man3~GlcNAc.Fuc~GlcNAcoT and Ga12-Fuc2.GlcNAc,. Man3.GlcNAc.FuoGlcNAcoT,respectively. For confirmation of the presence of a-fucosyl residues in the outer chain moieties of these oligosaccharides, radioactive N4-2 and N4-3 were incubated with almond a-fucosidase I. As shown in Figure 4H, N4-2 released one and N4-3 released two fucose residues by this treatment. The mobilities of radioactive products from

192

BIOCHEMISTRY

MATSUMOTO, YOSHIMA, A N D KOBATA

Table I: Methylation Analysis of N4-lb, N4-lu, N4-2, N4-3, and the Core Portion of N4-lb molar ratio N4-lba partially methylated sugars

N4-3 (-2Gal and -2GlcNAc)

N4-lb

N4- l~

N4-2

1.3

1.4

2.3

3.3

1.0

2.4

2.3

2.3

2.2

0

fucitol

2,3,4-triU-methyl(l,5-diU-acetyl) galactitol

2,3,4,6-tetra-O-methyl(l,5-di-O-acetyl) mannitol

2,3,4,6-tetra-O-methyl(l,S-di-O-acetyl) 0 0 0 0 2.3 2.2 2.4 2.1 3,4,6-tri-O-methyl(1,2,5-tri-O-acetyl) 1.0 0 1.0 1.0 2,4-di-O-methyl(1,3,5,6-tetra-O-acetyl) 2-monoU-methyl(1,3,4,5,6-penta-O-actyl) 0 1.0 0 0 2-(N-methylacetamido)-2-deoxygluci to1 0 0.6 0 0 3,4,6-triU-methyl(1,5-di-O-acetyl) 2.3 3.3 1.3 1.4 3,6-di-O-methyl(l,4,5-tri-O-acetyl) 1.2 0 1.1 0 4,6-di-O-methyl(l,3,5-triU-acetyl) 0.6 1.5 6-mono-O-methyl(l,3,4,5-tetra-O-acetyl) 0 0 0.9 0.7 0.8 0.7 1,3,5-triU-methy1(4,6-di-O-acetyl) a The solid line peak in Figure 4C. Numbers in the table were calculated by making the italicized values as 1.0.

2.3 0 I.0 0

0

1.3 0 0 0.9

a-fucosidase. These results indicated that N4-2 and N4-3 have one and two Gal@1-.(3- or -4)[Fuca1+(4- or -3)IGlcNAc groups in their outer chain moieties, respectively. Both radioactive products in Figure 4H gave exactly the same degradation pattern as N4- 1b (Figure 4@-G) by sequential exoglycosidase digestion. Therefore these oligosaccharides should also have the structure Gal@[email protected]([email protected]@ 1 4 M a n a1 4 3 )Man@l-4GlcNAc/3 1-+4(Fucal-+6)GlcNAcoT. The structural differences included in these three oligosaccharides were revealed by the following experiments. When incubated with a mixture of diplococcal @-galactosidase and diplococcal @-N-acetylhexosaminidase,the defucosyl N4-3 was converted to Man3-GlcNAc.Fuc~GlcNAcoT,but N4-lb and the defucosyl N4-2 were converted to a radioactive component with the same mobility as authentic Gal. GlcNAc~Man3~GlcNAc.Fuc~GlcNAcoT (Figure 41). These G results indicated that the two outer chains of defucosyl N4-3 are Gal@l-+4GlcNAc while one of the two outer chains of N4-lb and defucosyl N4-2 are either [email protected] or Gal@l-+6GlcNAc. The methylation analysis in Table I ini'; / \ I ;,>, 1 \ dicated that both N4-lb and N4-2 have 1 mol of C-3 sub300 400 500 stituted N-acetylglucosamine but not C-6-substituted N acetylglucosamine. Therefore, these oligosaccharides should ELUTION VOLUME (ma) have one each of Gal@l-+4GlcNAc and Gal@l-+3GlcNAc FIGURE 4: Sequential exoglycosidase digestion of oligosaccharides groups in their outer chain moieties. The data in Table I also N4-lb, N4-2, and N4-3. Black arrows are the same as in Figure 3. White arrows indicate the eluting positions of authentic oligoindicate that both N4-2 and N4-3 have all of their galactose saccharides: I, Ga12~Fuc2~GlcNAc2~Man3~GIcNA~Fu~GlcNAc~T; and fucose residues as nonreducing termini and N4- 1b, N4-2, 11, Ga12~Fuc.GlcNAc2~Man3-GlcNA~Fuc~GlcNAc~T; 111, Gal2. and N4-3 have one, two, and three fucoses, respectively. GlcNAc2~Man3~GlcNAc~Fuc~GlcNAc~~; IV, GlcNAc2.Man3Detection of 6-mono-O-methyl-2-(N-methylacetamido)-2G~cNAc.FuQG~cNAc~T; V, Gal.GlcNAc-ManS.GlcNAc.Fuc. GlcNAcoT; VI, Man3~GlcNAc~Fuc~GlcNAcoT; VII, Man. deoxyglucitol in both N4-2 and N4-3 indicates that the fucose GlcNAc.Fuc.GlcNAcoT; VIII, G~cNAoFuoG~cNAcoT; IX, FUO residues in the outer chain moieties of these oligosaccharides GICNACOT;X, N-acetyl[3H]glucosaminitol.(A) Oligosaccharides occur as Gal@l-+4(Fucal-+3)GlcNAc groups. Detection of N4-lb (-), N4-2 (---),and N4-3 (-*-); (B) oligosaccharides N4-lb approximately 2 mol of 3,4,6-tri-Qmethylmannitol in all three (-) N4-2 (- - -), and N4-3 (- --) incubated with jack bean @-galacoligosaccharides indicates that their two outer chain moieties tosidase; (C) oligosaccharides in (B) derived from N4-lb (-) and N4-2 (---) incubated with jack bean @-N-acetylhexosaminidase;(D) are linked at the C-2 positions of two a-mannosyl residues of oligosaccharidein (C) derived from N4-lb incubated with jack bean the trimannosyl core. On the basis of the results so far dea-mannosidase; (E) the radioactive peak in (D) incubated with scribed, the structures of N4-lb, N4-2, and N4-3 were pro@-mannosidase;(F) the radioactive peak in (E) incubated with jack posed as shown in Figure 8. That the Gal@l+3GlcNAc bean @-N-acetylhexosaminidase; (G) the radioactive peak in (F) incubated with Charonia Zampas a-fucosidase; (H) oligosaccharides groups in N4-lb and N4-2 are located on the Manal-3 side N4-2 (- - -) and N4-3 (- -) incubated with almond a-fucosidase I; was confirmed as follows. When the radioactive components (I) oligosaccharides in (H) derived from N4-2 (- - -) and N4-3 (- -) shown in Figure 41 obtained from N4-lb and N4-2 were and intact N4-lb (-) incubated with a mixture of diplococcal @incubated with jack bean a-mannosidase, no mannose residue galactosidase and diplococcal @-N-acetylhexosaminidase. was removed from both oligosaccharides (data not shown). both oligosaccharides were slightly different. No degradation Since this enzyme releases a mannose residue from R+occurred when N4-2 and N4-3 were incubated with Bacillus Mana 14 6 ( Mana 1-+3)Man@ 1-4GlcNAc-but not from

.

VOL. 22, N O . 1 , 1983

CARBOHYDRATES OF INFLUENZA VIRUS

I

1 7 1 6 15 1 4 13 12

vo

11

IO

9

8

1

vo

7

1

350 300

1

0

1

400

9

1

8

7

1

6

1

450

ELUTION VOLUME

400

ELUTION VOLUME (me)

FIGURE 6:

5

4

3

1

1

1

500

193

550

(me)

Periodate oxidation products of oligosaccharide N4- lu.

For details of the periodate oxidation see Experimental Procedures. The black arrows are the same as in Figure 3. (A) The products from radioactive oligosaccharide N4- lu; (B) the products from authentic

FIGURE 5: Sequential exoglycosidase digestion of oligosaccharide N4-lu. Black arrows are the same as in Figure 3. White arrows Ga12~GlcNAc2~Man3~G1cNAc.Fuc.GlcNAc~~. indicate the eluting positions of authentic oligosaccharides: I, Ga12~GlcNAc2~GlcNAc~Man3~GlcNAc~Fuc~GlcNAcoT; 11, GlcNAc2-GlcNAc-Man3.GlcNAc.Fuc.GlcNAcOT; 111, Man3. GlcNAcp1+4Man@l+ group (Figure 6B). Therefore, the GlcNAc.Fuc.GlcNAcoT. (A) Oligosaccharide N4- 1u; (B) oligoGal@l+4GlcNAc group should not be linked at the C-4 saccharide N4-lu incubated with jack bean /3-galactosidase; (C) the position of the P-mannosyl residue because such a structure radioactive peak in (B) incubated with jack bean 8-N-acetylhexosaminidase. would produce the GlcNAc@l+4Man@l+ group after periodate oxidation. On the basis of the results so far described, Mana 1+6( R-Mana 1-.3)Man@l+4GlcNAc(Y amashita the structure of N4-lu is proposed as shown in Figure 8. et al., 1980b), the Gal@[email protected] outer chains in the Structures of N4-4, N4-5, N4-6, and N4-7 Fractions. Upon two radioactive oligosaccharides should be linked only to the Bio-Gel P-4 column chromatography, N4-4, N4-5, and N4-6 Manal-3 residue. were eluted as single peaks and the mobilities of N4-4 and Upon Bio-Gel P-4 column chromatography, N4-lu was N4-6 were the same as those of authentic Ga13.GlcNAc3. eluted as single peak with the same mobility as authentic Man3~GlcNAc~Fuc~GlcNA~oT and Ga14.GlcNAc4.Man3. Ga12GlcNA~GlcNAc~Man3~GlcNAc.Fuc.GlcNAc~ (Figure G~cNAc*Fuc*G~cNAc~, respectively (Figure 7A). In conSA). When incubated with jack bean @-galactosidase,it was trast, N4-7 was separated into two components as indicated converted to a radioactive component with the same mobility by bars I and I1 in Figure 7A. These two components were as authentic GlcNAc2~GlcNAc~Man3~GlcNAc.Fuc~GlcNAcm collected separately and named as N4-71 and N4-711, re(Figure 5B). It was then converted to a radioactive component spectively. with the same mobility as authentic Man3-GlcNAc.FucWhen incubated with almond a-fucosidase I, N4-5 and GlcNAcoT by jack bean P-N-acetylhexosaminidasedigestion N4-71 released 1 mol and N4-711 2 mol of fucose residue (Figure 5C). That this component actually has the structure (Figure 7B). The product from N4-5 gave exactly the same (Mana l-.)2Man@l +GlcNAc@l-.(Fuccul +)GlcNAcm was results as N4-4 and the products from N4-71 and N4-711 as confirmed by further sequential exoglycosidase digestion, the N4-6 by the structural analyses described below. data of which were completely the same as shown in Figure When incubated with jack bean 0-galactosidase, 3 and 4 4D-G. Methylation analysis of N4-lu in Table I indicates mol of galactose were removed from N4-4 and N4-6 (Figure that the oligosaccharide has the following hexasaccharide as 7C). The products from N4-4 and N4-6 were then converted its core to which a @-N-acetylglucosamineresidue and two to a radioactive component with the same mobility as authentic Gal@l+4GlcNAcfll- groups are linked. Man3~GlcNAc.Fuc.GlcNA~oT releasing 3 and 4 mol of Nacetylglucosamine by jack bean O-N-acetylhexosaminidase Fucal digestion, respectively (Figure 7D). That the products from -2Manol 1 c‘ ” 4 N4-4 and N4-6 at this stage have the structure Manal-6Man P 1 -4GlcNAcP 1 4GlcNAco, (Mana 13’) Man@1-4GlcNAc@1+4( Fuca 1--6)GlcNAcoT / was confirmed as in the case of the solid line peak in Figure -2Manal 4C. The sequential exoglycosidase digestion so far described indicates that the structure of N4-4 can be written as That the 8-N-acetylglucosamine residue is exclusively linked (Gal@l+GlcNAc@ 1+)3 [ Mana 1+6( Mana 1+3)Man@l+to the C-4 position of the P-mannosyl residue was confirmed 4GlcNAc@1+4(Fucoll+6)GlcNAcoT] and that of N4-6 as by periodate oxidation. When the periodate oxidation product (Gal@l+GlcNAc@l+)4[ Mana 1+6( Mana 1+3)Manp 1-+of radioactive N4- 1u was analyzed by Bio-Gel P-4 column, 4GlcNAc@1+4( Fucal+6)GlCNACoT]. two radioactive components were detected (Figure 6A). The The methylation analysis of N4-4 in Table I1 indicated that major component should be Man@[email protected] because it was converted to N - a ~ e t y l [ ~ H ] - a Galpl+4GlcNAc@l+ group is linked at the C-2 position of one a-mannosyl residue and two Galpl+4GlcNAc01-+ glucosaminitol by sequential digestion with @-mannosidaseand groups are linked at C-2 and C-4 positions of another ajack bean @-N-acetylhexosaminidase(data not shown). The mannosyl residue of the core portion. For determination of minor component is considered as a byproduct and not which of the two a-mannosyl residues is C-2,4 disubstituted, GlcNAc@[email protected]@l+4GlcNAcm because it radioactive N4-4 was incubated with a mixture of diplococcal was converted to a radioactive component which moved faster @-galactosidaseand diplococcal 0-N-acetylhexosaminidase. than Man~l+4GlcNAc@l+4GlcNAcm by 2.0 glucose units When the reaction mixture was analyzed by a Bio-Gel P-4 in the Bio-Gel P-4 column after @-N-acetylhexosaminidase column, a single radioactive component with the same mobility digestion. This minor component was also detected in the as authentic GlcNAc.Man3~GlcNAc.Fuc.GlcNAcoT was deperiodate oxidation product of authentic Ga12.GlcNAc2. tected (Figure 7E). On the basis of the substrate specificity Man3.GlcNAc.Fuc.GlcNAcoT which does not contain the

-

194

MATSUMOTO, YOSHIMA, A N D KOBATA

BIOCHEMISTRY

Table 11: Methylation Analysis of N4-4, N4-5, N4-6, and N4-7 molar ratioa partially

methylated sugars

N4-4

N4-5 N4-6

N4-7

fucitol

2,3,4-tri-O-methyl (1,5-di-O-acetyl) galactitol 2,3,4,6-tetraU-methyl (1,5-di-O-acetyl) mannitol

0.9

2.2

1.1

2.5

3.2

3.5

4.4

4.5

3,4,6-tri-O-methyl

1.2

1.4

0

0

(1,2,5-tri-O-acetyl) 3,6-di-O-methyl

0.7

0.7

0.8

0.9

0

0

0.6

0.6

1.0

1.0

1.0

3.5

5.3

3.7

0.6

0

1.3

(1,2,4,5-tetra-O-acetyl)

3,4-di-O-methyl (1,2,5,6-tetra-O-acetyl)

1“

I

1.0 2,4-di-O-methyl (1,3,5,6-tetraU-acetyl) 2-(N-methylacetamido)2-deoxyglucitol 3,6-di-O-methyl 4.4 (1,4,5-tri-O-acetyl) 6-mono-0-methyl 0

\

G

n

I

300

ELUTION VOLUME

400

(1,3,4,5-tetra-O-acetyl)

1,3,5-tri-O-methyl 1.3 1.2 1.3 1.3 (4,6-di-O-acetyl) Numbers in the table were calculated by making the italicized values as 1.0.

(me)

FIGURE 7: Sequential exoglycosidase digestion of oligosaccharides N4-4, N4-5, N4-6, and N4-7. Black arrows are the same as in Figure 3. White arrows indicate the eluting positions of authentic oligosaccharides: I, Ga14~GlcNAc4~Man3~G1cNAc.Fuc.GlcNAcoT; 11, Ga13~GlcNAc3~Man3~G1cNAc.Fuc.GlcNAcoT; 111, GlcNAc,.Man,. GlcNAc-Fuc.GlcNAw; IV, GlcNAc3~Man3GlcNAc.Fuc.GlcNA~;hexosaminidase, 2 mol each of galactose and N-acetylglucosamine was removed from N4-5 [Figure 7G,H (- - -)I, V, GlcNAc.Man3.GlcNAc.Fuc.GlcNAcoT; VI, Man,.GlcNAc. Fuc.GlcNAcoT. (A) Oligosaccharides N4-4 (-), N4-5 (- - -), N4-6 respectively. The radioactive product in Figure 7H (-- -) was (-.-), and N4-7 (-); (B) oligosaccharides N4-5 ( - - - ) N4-71 completely resistant to jack bean a-mannosidase digestion, and N4-711 (--) incubated with almond a-fucosidase; (C) oligoindicating that the remaining Galpl-.4(Fuca1-+3)saccharides N4-4 (-) and N4-6 (-.-) incubated with jack bean GlcNAcPl- outer chain is linked only to Manal-3 side. A @-galactosidase;(D) the radioactive peaks in (C) incubated with jack bean @-N-acetylhexosaminidase;(E) oligosaccharideN4-4 incubated fucose residue and a galactose residue were removed from the with a mixture of diplococcal @-galactosidaseand diplococcal @-Nproduct in Figure 7H by sequential digestion with almond acetylhexosaminidase; (F) the radioactive peak in (E) incubated with a-fucosidase I and jack bean @-galactosidase(data not shown). jack bean 8-N-acetylhexosaminidase;(G) oligosaccharides N4-5 (-- -), The resulting product was completely resistant to diplococcal N4-71(...), and N4-711 (-) incubated with jack bean @-galactosidase; P-N-acetylhexosaminidasetreatment. These results indicated (H) the radioactive peaks in (G) incubated with jack bean @-Nacetylhexosaminidase. that the N-acetylglucosamine residue of the Gal@1+4(Fucal+3)GlcNAc~l-+ group should exclusively be linked at the C-4 position of the Mancul-3 residue. On the basis of diplococcal P-N-acetylhexosaminidase(for details see Experimental Procedures), the structure of the radioactive comof a series of results described above, the structure of N4-5 is proposed as shown in Figure 8. ponent in Figure 7E was concluded to be GlcNAcPl+As already described, N4-71 and N4-711 were mono- and 4Manal-(3or -6)[Mana1+(6- or -3)]Manpl+difucosyl derivatives of N4-6. Because the fucosyl residues 4GlcNAcpl-+4(Fuca 1-6)GlcNAcoT. Actually the radioactive component in Figure 7E was converted to Mancul-6could be removed by almond a-fucosidase I, they should occur as the Gal~l-4(Fuca1-3)GlcNAc~1- group. Detection (Mana 1-3) Mano 1+4GlcNAc@ 1+4( Fuca 1-6)GlcNAcoT by jack bean P-N-acetylhexosaminidasedigestion releasing 1 of 6-mono-0-methyl-2-(N-methylacetamido)-2-deoxyglucitol mol of N-acetylglucosamine (Figure 7F). When the radioin the methylation data of the N4-7 fraction (Table 11) supactive component in Figure 7E was incubated with jack bean ports this assumption. a-mannosidase, no degradation occurred (data not shown). When incubated sequentially with jack bean P-galactosidase This result indicated that an N-acetylglucosamine residue and jack bean P-N-acetylhexosaminidase, 3 mol each of galactose and N-acetylglucosamine was removed from N4-71 and should be linked exclusively to the Manal-3 side. Therefore, the structure of N4-4 is proposed as shown in Figure 8. 2 mol each of galactose and N-acetylglucosamine from N4-711 Methylation analysis of N4-6 indicated that two GalPl-+[Figure 7G,H (.-)I. The radioactive products in Figure 7H 4GlcNAc/31- groups are linked at the C-2 and C-4 positions from N4-71 and N4-711 were completely resistant to jack bean of one a-mannosyl residue and two G a l ~ l - 4 G l c N A c ~ l + a-mannosidase digestion. Therefore, the Gal(31-.4(Fuccul+groups at the C-2 and C-6 positions of the other a-mannosyl 3)GlcNAcPl- group of N4-71 and at least one of the two residue of the trimannosyl core as shown in Figure 8. The Gal0 1+4( Fuca 1-3)GlcNAcP 1 groups of N4-711 should exact location of C-2,4 and C-2,6 disubstitution could not be be linked to the Manal-3 side of the trimannosyl core. A determined because of the limited amount of sample available. fucose and a galactose were removed from peak I in Figure As already described, N4-5 is a monofucosyl derivative of 7H by sequential digestion with almond a-fucosidase I and N4-4, and the fucose residue should be linked at the C-3 jack bean &galactosidase (data not shown). The radioactive position of the N-acetylglucosamine residue of one of the three heptaitol thus obtained was converted to Manal-6Gal~l+4GlcNAc~l-+outer chains of N4-4. When incubated (Mana 1-3)Man(31-4GlcNAcP 1+4( Fuca 1-6)GlcNAco~ sequentially with jack bean @galactosidase and 0-N-acetylby jack bean P-N-acetylhexosaminidase digestion but was (e-),

+

VOL. 22, NO. 1 , 1983

CARBOHYDRATES O F INFLUENZA VIRUS N1

(Manal-).) *ManBl+GlcNAc61 *GlCNACOT

Fucal

+

(9.8)

N2 (1 3.9)

Manal+Z[(Manal+)r~Man61*GlcNAc61*G1CNACoT]

N3-1 (34.5)

GlcNAc51+2 [ (Manal+) ~Man61*GlcNAc61*GlcNAcoT]

N3-2 (15.2)

{Manal+2) [ {Manal-.) ~ManB1-ClcNAcB1*GlcNAcOT]

I

(8.8) b

N4-1

195

Gal 61 *4GlcNAc 61 * 2 M a n a h 6 +Man61 Gal 51 *3GlcNAc 61 *ZManal

N4-5 (2

'

N4-6 *4R

(2*3)

3 Gal 61 *4GlcNAc 5

1 ~ -8 M a n a k 3 Gal 5 1 +4Gl~NAc61'~ +4R -161 +4GlCNAC51 * 2 M a n a 4 M a n B 1

-

Gal 6 1 +4GLCNACB k A lanal Galgl+4GlcNAcB@ 3 ' Galg1-4GlCNAcg

or %an61+4R

GlcNAcBl i

Gal61 +4GlcNAc 81 *ZManal\ 6

4 Fucal

GalB1*4GlcNA~61+2Manal~~~~~~*~~

).

FUCQ~

N4-71

3 G a l ~ 1 + 4 G l C N A c B 1 ~Of 4

).

N4-2 (2.2)

3 Gal ~ 1 + 4 G l c N A c B 1 + 2 M a n a 1 ~ Man51*4R Gal 51 +3Gl~NAc51+2ManaY'~

'O

Or

Fucal

).

N4-3 (4.2)

Ga 1 6 1 +4G 1cNAC 5 1 *2Manal h6 Mangl +4R Gal 5 1 t4GlcNAc 51 *ZMana 3 t Fucal

Manu 1 "ManBl+4R 4Manap

Ga 16 1 + 4GlcNAc 5"1

Fucal 3

Ga151*4GlCNAC51\ Gal 5 1+4Gl~NAc51/~

N4-711

+

Gal51*4GlcNAcBl 3

zManalh6 3Man61*4R

(Oe8)

(Gal~1-4GlcNAc51)I 2 M a n a l R 3

Or

Fucal N4-4 (1.5)

GalB1+4GlcNAcBk,

+

R=GlcNAcB1*4GlcNAc0T 6

Gal51 + ~ G ~ C N A C ~Man51 P ~ +4R ~ " ~ Gal 5 1 + 4 G l c N A c B 1 * 2 M a n a ~

Structures of neutral oligosaccharides obtained from X-3 1 BHA glycoprotein. Numbers in parentheses are percentage molar ratio of the sugar chains in BHA.

FIGURE 8 :

totally resistant to diplococcal @-N-acetylhexosaminidase treatment (data not shown). These results indicate that the [email protected](Fuca1-+3)GlcNAc@1-. group of N4-71 is linked to either the C-4 or C-6 position of a-mannosyl residue as shown in Figure 8. So far, the exact location of the two [email protected](Fucal-+3)GlcNAcfll- groups in N4-711 has not been determined. Discussion The complete amino acid sequence of the HA of X-31 strain of A2/Hong Kong/1968 virus was elucidated by Ward & Dopheide (1981) and by Verhoeyen et al. (1980). The data indicated that the HA contains seven asparagine-linked sugar chains. These sugar chains are attached at asparagine residues 8, 22, 38, 81, 165, and 285 in the HA1 subunit and 154 in the HA2 subunit, and the sugar chain on asparagine-8 of HA1 subunit is supposed to contain a sulfate residue. Several works concerning the carbohydrate moieties of HA have been reported (Schwarz & Klenk, 1981; Ward & Dopheide, 1981). However, the information reported in these papers was limited because it was based either on the monosaccharide compositions of glycopeptides obtained by cyanogen bromide fragmentation or on gel permeation analysis of the glycopeptides obtained by exhaustive Pronase digestion of the metabolically labeled HA with radioactive monosaccharides. Because BHA contains most of the sugar chains of intact HA, the results reported in this paper may represent most of the characteristics of the sugar chains of HA. Approximately 20% of the sugar chains of BHA was acidic. This result indicates that more than a single sugar chain in BHA might be sulfated. However, this conclusion must be refrained until more structural information on the acidic sugar chains will be obtained, because the action of sulfatase on the acidic sugar chains was far from quantitative. The structures of almost

all of the neutral sugar chains of BHA were elucidated in this paper. The results summarized in Figure 8 indicate that the sugar chains of influenza virus hemagglutinin are formed by the processing pathway (Kornfeld & Kornfeld, 1980) because N1, N2, N3-1, and N3-2 accord with the processing intermediates. Structures of the remaining oligosaccharides, however, indicate that these sugar chains are formed by complicated routes. N4-lb and N4-2 should be formed by the pathway different from that forming other sugar chains. In addition, N4-lu, N4-3, N4-5, and N4-711 are considered as final products in their biosynthetic route. It is particularly noteworthy that only N4-lu among the complex type sugar chains contains the N-acetylglucosamine residue linked to p-mannose. In other glycoproteins which have this bisectional N-acetylglucosamine in their sugar chains, the N-acetylglucosamine residue is more widely distributed in their sugar chains. The oligosaccharide pattern of BHA reported here is somewhat different from those of HANA protein and F protein, the two envelope glycoproteins of HVJ which were also grown in the allantoic sac of embryonated hen eggs (Yoshima et al., 1981). The two glycoproteins of HVJ do not contain any tri- and tetraantennary complex type sugar chains. The complex type sugar chains with the Gal@l+3GlcNAc group in their outer chain moieties (N4-lb and N4-2 in Figure 8) and with an N-acetylglucosamine residue linked to @mannose (N4-lu in Figure 8) are also absent in the two glycoproteins of HVJ. These differences may indicate that the sugar chains of envelope glycoproteins of the two viruses are not simple replicas of host membrane glycoproteins as suggested in the case of vesicular stomatitis virus (Hunt, 1980). In Figure 8, the percent molar ratio of each sugar chain in the BHA molecule is also included. One of the prominent features is that the sum total of the complex type sugar chains is only 26.6% of all neutral sugar chains. Therefore the

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complex type sugar chains can occupy at most two asparagine residues if they are attached at specific asparagine residues as suggested by several investigators. This estimation does not accord with the analytical result reported by Ward & Dopheide (1981) which showed that five asparagine loci of X-3 1 influenza hemagglutintin are occupied by complex type sugar chains. A possible explanation for this discrepancy is that many asparagine loci are substituted by both the complex type and the high mannose type sugar chains. The glycopeptide fragments containing such loci might have been concluded to have complex type based only on their monosaccharide compositions. The possibility that the sugar chains were modified during bromelain treatment of H A to obtain BHA is rather improbable but cannot be completely denied at the present stage. Therefore, a comparative study of the oligosaccharide patterns of glycopeptide fragments with a single asparagine-linked sugar chain from HA is essential to confirm the above estimation. Acknowledgments We thank Dr. John Skehel, National Institute for Medical Research, London, for kindly supplying us the BHA sample and for his help in making this paper. Thanks are also due to the skillful secretarial assistance of Ikuko Ueda. Registry NO. N1,83816-30-2; N2,83816-31-3; N3-1,83844-72-8; N3-2, 83830-93-7; N4-1 b, 83830-94-8; N4-1 U, 83816-32-4; N4-2, 83816-33-5; N4-3,83368-45-0; N4-4, 80968-39-4; N4-5,83830-95-9.

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